Dengue fever is a major public health concern, including 185,000 annual cases in Cambodia. Aedes aegypti is the primary vector for dengue transmission and is targeted with insecticide treatments. This study characterized the insecticide resistance status of Ae aegypti from rural and urban locations. The susceptibility to temephos, permethrin, and deltamethrin of Ae aegypti was evaluated in accordance with World Health Organization instructions. All the field populations showed lower mortality rate to temephos compared with the sensitive strain with resistance ratio 50 (RR50) varying from 3.3 to 33.78 and RR90 from 4.2 to 47 compared with the sensitive strain, demonstrating a generalized resistance of larvae to the temephos in Cambodia. Ae aegypti adult populations were highly resistant to permethrin regardless of province or rural/urban classification with an average mortality of 0.02%. Seven of the 8 field populations showed resistance to deltamethrin. These results are alarming for dengue vector control, as widespread resistance may compromise the entomological impact of larval control operations. Innovative vector control tools are needed to replace ineffective pesticides in Cambodia.